Journal
JOURNAL OF STRUCTURAL BIOLOGY
Volume 209, Issue 1, Pages -Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.jsb.2019.10.004
Keywords
Microtubule; Pseudo-helical symmetry; Cryo-EM; RELION; 3D reconstruction
Funding
- Medical Research Council, U.K. [MR/R000352/1, MR/J003867/1]
- Wellcome Trust [079605/Z/06/Z, 101488/Z/13/Z]
- BBSRC [BB/L014211/1]
- MRC [MR/R000352/1] Funding Source: UKRI
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Microtubules are polar filaments built from alpha beta-tubulin heterodimers that exhibit a range of architectures in vitro and in vivo. Tubulin heterodimers are arranged helically in the microtubule wall but many physiologically relevant architectures exhibit a break in helical symmetry known as the seam. Noisy 2D cryo-electron microscopy projection images of pseudo-helical microtubules therefore depict distinct but highly similar views owing to the high structural similarity of alpha- and beta-tubulin. The determination of the alpha beta-tubulin register and seam location during image processing is essential for alignment accuracy that enables determination of biologically relevant structures. Here we present a pipeline designed for image processing and high-resolution reconstruction of cryo-electron microscopy microtubule datasets, based in the popular and user-friendly RELION image-processing package, Microtubule RELION-based Pipeline (MiRP). The pipeline uses a combination of supervised classification and prior knowledge about geometric lattice constraints in microtubules to accurately determine microtubule architecture and seam location. The presented method is fast and semi-automated, producing near-atomic resolution reconstructions with test datasets that contain a range of microtubule architectures and binding proteins.
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